J. clin. Path., 1976, 29, 601-607
Significance of urinary C3 excretion in glomerulonephritis A. D. CUMMING, D. THOMSON, A. M. DAVISON, AND J. S. ROBSON From the Department of Pathology and the Medical Renal Unit, Department of Medicine, University of Edinburgh and the Royal Infirmary, Edinburgh EH3 9 YW
The third component of complement (C3) was measured in the urine of 98 patients with variety of renal diseases. Renal biopsy was performed on 83 of the patients and examined by light, electron, and immunofluorescence microscopy. Urinary C3 was detected in cases of membranous glomerulonephritis, mesangiocapillary glomerulonephritis, rapidly progressive glomerulonephritis, and renal amyloidosis. It was not detected in minimal lesion glomerulonephritis; in cases of proliferative glomerulonephritis it was detected only in those showing histological evidence of a progressive lesion. Concentrations were low or undetectable in cases of non-immunological renal diseases. There was a good correlation between urinary C3 concentrations and the deposition of C3 in glomerular capillary walls, as seen by immunofluorescence microscopy, and there was no correlation with the degree or selectivity of proteinuria. Urinary C3 excretion appears to be an accurate indicator of continuing activity of disease. It is suggested that the presence of C3 in urine is due to complement fixation by immune complexes in glomerular capillary walls, and that urinary C3 estimations have potential applications in the study of glomerulonephritis.
SYNOPSIS a
The association of complement with renal disease has been known since 1902 when Hedinger described low serum complement in 'acute nephritis'. Many studies have subsequently shown that low serum complement titres are characteristic of acute post-
information about renal immune processes. Lange and Wenk (1954) investigated urinary loss of complement as a possible cause of low serum whole complement titres in glomerulonephritis and found that there was no correlation between the urinary streptococcal glomerulonephritis, mesangiocapillary complement titre and the serum complement titre, glomerulonephritis, systemic lupus erythematosus or the degree of proteinuria. Gotoff et al (1965) with renal involvement, and nephritis accompanying found C3 in the urine of patients with nephrotic subacute bacterial endocarditis (Lange et al, 1951, syndrome with heavy proteinuria but not in cases 1960; West et al, 1964; Gotoff et al, 1965; Ogg et al, of acute or progressive glomerulonephritis. Ogg et al 1968; Gutman et al, 1972). This is thought to be (1968) found urinary C3 in only one of 25 patients due in many cases to utilization of complement by with low serum C3 concentrations. immune complexes (Kohler and Ten Bensel, 1969; Lagrue et al (1969) examined complement activity Koffler et al, 1971), although direct activation of the in the urine of 24 patients with glomerulonephritis third component by the alternate pathway is known and found complement components in the urine of to occur in some types of glomerulonephritis nine patients. Complement excretion did not (Vallota et al, 1970). It has also been shown that in correlate with the degree or selectivity of proteinuria many situations there is a correlation between the but varied in the four types of glomerulonephritis whole complement titre and the concentration of studied. They concluded that the mechanism of the third component of complement (C3) (Klem- urinary complement excretion remained to be determined and that its frequency and significance perer et al, 1965; Lundh et al, 1970). Since complement is activated during immune should be further studied. Hoq et al (1974) studied reactions, it seems possbile that examination of 15 cases of proliferative glomerulonephritis and 10 complement excretion in the urine could give cases after renal homotransplantation. Urinary C3 concentrations correlated with urinary concentrations of fibrin degradation products and heteroReceived for publication 3 December 1975 601
602
A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
phile haemagglutinins, were not related to total urinary protein, and gave information on response to treatment and occurrence of rejection. Williams et al (1974) studied 123 patients with glomerulonephritis; urinary C3 was detected often in mesangiocapillary and membranous glomerulonephritis and in focal glomerulosclerosis, less frequently in lupus nephritis and a mixed group of cases with proliferative glomerulonephritis, and rarely in minimal lesion glomerulonephritis. The present study examines the concentration and significance of C3 in the urine of patients with renal disease. Immunofluorescence microscopy has demonstrated the presence of material, consisting of immunoglobulins and complement components, within the glomerular capillary wall in several forms of glomerulonephritis, and it seems likely that deposition of such material could give rise to increased components of complement in the urine. This was investigated by examining (1) the relationship of urinary C3 to the type of glomerulonephritis present, and also urinary C3 concentrations in cases of proteinuria thought not to be due to acomplementfixing glomerular immune reaction; (2) the relationship between urinary C3 concentrations and C3 deposition in glomerular capillary walls detected by immunofluorescence microscopy; (3) the relationship between urinary C3 concentrations and the degree and selectivity of proteinuria.
and a raised ASO titre. In 11 cases there were no polymorphs in the glomeruli, there was slight to moderate hypercellularity of mesangial regions, and these were considered to be cases of mild proliferative glomerulonephritis. Twenty-two cases showed enlarged, hyaline, and hypercellular mesangial regions, producing variable degrees of hyalinization of the tufts, and in some cases capsular adhesions and small epithelial crescents were additional features of note. These changes were considered to indicate progression of the disease process resulting in some permanent glomerular damage. They were therefore designated as cases of proliferative glomerulonephritis showing histological evidence of progression. There were six cases of rapidly progressive glomerulonephritis, the main histological features being florid circumferential epithelial crescents in most glomeruli, compressing the capillary tufts, with little or no mesangial hypercellularity. Other types of renal disease were also diagnosed histologically according to conventional criteria, except for eight cases of non-immunological renal disease and seven cases of urinary tract infection, which were diagnosed on clinical grounds since a biopsy had not been performed. HISTOLOGICAL METHODS
All biopsies were examined by light microscopy; electron microscopy was performed in 64 cases and immunofluorescence microscopy in 51. For light Material and Methods microscopy, biopsy material was fixed in corrosive formol saline for 18-24 hours, sectioned at 2 ,um, PATIENTS STUDIED The concentration of urinary C3 was measured in and stained by the haematoxylin and eosin, picro 98 patients, 83 of whom underwent renal biopsy. Mallory, periodic-acid Schiff, and Martius scarlet The histological diagnoses are shown in the table. blue methods. For electron microscopy the tissue Of the 18 cases of proliferative glomerulonephritis was fixed in 1 % osmium tetroxide, sometimes with without histological evidence of progression, seven preliminary glutaraldehyde fixation. It was dehydshowed an acute, exudative lesion. They were rated in graded alcohols and embedded in Araldite histologically similar to classical poststreptococcal or Epon. Sections were cut on a Porter Blum MT2 glomerulonephritis, showing diffuse enlargement of ultratome at 50 nm, stained with lead citrate and tufts, moderate to marked mesangial hypercellularity, uranyl acetate, and viewed in an AEI EM6 electron and a polymorphonuclear infiltrate. Two of these microscope. For immunofluorescence microscopy cases had a proven previous streptococcal infection fresh tissue was 'snap frozen' with Drikold and sectioned at 2 to 3 ,um in a cryostat. After fixation with 95 % alcohol for 10 minutes, sections were No. of Cases Type of Disease incubated with fluorescein isothiocyanate conjugated (FITC) antihuman serum to C3, IgG, IgA, IgM, and Proliferative glomerulonephritis 18 no histological evidence of progression fibrin-fibrinogen (Hoechst). Sections were viewed 22 histological evidence of progression in a Leitz Ortholux microscope using an HBO 200 6 Rapidly progressive glomerulonephritis 15 Membranous glomerulonephritis with BG12 and BG38 primary filters and a lamp 9 Mesangiocapillary glomerulonephritis K510 barrier filter. 5 Minimal lesion glomerulonephritis Renal amyloidosis Non-immunological renal disease Urinary tract infection
4 12 7
Table Diagnoses and number ofpatients of each type
URINARY c3 AND SELECTIVITY
For C3 estimations 10-30 ml samples of urine were used; where possible these were early morning
Significance of urinary C3 excretion in glomerulonephritis
603
obtained. Four serial dilutions of a pool of 30 normal human sera were included on each plate. The C3 concentration of this pool was measured using standard sera (Travenol Laboratories) and found to be 1-3 g/l. Results of test samples were expressed as mg/l. A single sample was tested on 20 separate occasions and the standard deviation was found to be ± 8 %. Protein selectivity was estimated by the method of MacLean and Robson (1967).
specimens. Three to four daily samples were obtained from most patients and the highest urinary C3 concentration found in each patient was used in subsequent analysis. Samples were tested for total protein, using Albustix (Ames), dialysed against running tap water for 8 hours, and left overnight in a solution of polyethylene glycol (Carbowax) at - 40C, which concentrated each sample 10-15 times. Samples were stored at -30°C until they were tested. C3 concentrations were measured by single radial immunodiffusion; plates were prepared using a modification of the method of Mancini (Mancini et al, 1965). 0 3 ml of rabbit antiserum to human /IC-PIA globulin (Hoechst) was used in each 16-well plate. We have not ascertained with which fragments of C3 the antiserum reacts; previous work suggests it may react with intact C3, C3b, and C3c (West et al, 1966; Bokisch et al, 1969; Ruddy et al, 1972). The antiserum was used in immunoelectrophoresis of aged human serum; a single arc was
Results RELATION
OF
URINARY
c3
TO HISTOLOGY
(FIGURE) Urinary C3 was not detectable in any of the 18 patients with proliferative glomerulonephritis who showed no histological evidence of progression. Of the seven patients in this group with an acute exudative lesion, all had moderate or heavy proteinuria and three showed immunofluorescence to C3 in glomerular capillary walls. Nine of the 11
TYPE OF RENAL DISEASE
Proliferative Glonierulonephritis - no histological evidence of progression
Proliferative Glonierulonephritis
-
histological evidence of progression
0'#
Rapidly Progressive Glomerulonephritis
.
s
Membranous Glomerulonephritis
1*00i *0 em
Mesangiocapillary Glomerulonephritis
*
0
C
0
*
I
me *
I
0
I
G*~~~~~~~~~~
Minimal Lesion Glomerulonephritis
00n Renal Amyloidosis
Non-Immunological Renal Diseases Urinary Tract Infection
10
0
20
30
40
Figure Maximum concentrations of C3 in urine from patients with renal disease. Conversion: SI to Traditional Units-C3: I
50
URINARY C3 CONCENTRATION * DETECTABLE NOT DETECTABLE
mg/li = 0.1 mg/100 ml
60
mg/l)
70
80 100
260
604
A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
patients with mild proliferative glomerulonephritis showed significant proteinuria and one showed some C3 deposition in glomerular capillary walls by immunofluorescence microscopy. In follow-up observations between 13 months and five years after onset of the disease, one of the 18 patients had raised plasma urea and creatinine concentrations, and one had recurrent proteinuria. The other patients were symptom-free, had normal plasma urea and creatinine concentrations, normal blood pressure, and no proteinuria or haematuria. In contrast, 21 of the 22 cases of progressive proliferative glomerulonephritis showed significant urinary C3 concentrations (median 10 mg/I, range 0-210 mg/i). Of the 12 cases studied by immunofluorescence microscopy, five showed immunofluorescence to C3 in glomerular capillary walls. In follow-up studies between one and 11 years after onset of the disease, all but one of the 22 patients showed one or more of the following abnormalitiesraised plasma urea or creatinine concentration, raised blood pressure, persistent proteinuria or haematuria. The patient whose disease clinically resolved had a maximum urinary C3 concentration of 10 mg/l. The six cases of rapidly progressive glomerulonephritis all showed detectable urinary C3, but, in comparison with the progressive proliferative cases, a smaller range of concentrations was found (median 12 mg/I, range 8-25 mg/i). Four cases showed immunofluorescence to C3 in glomeruli. The greatest range of urinary C3 concentrations was found in the 15 cases of membranous glomerulonephritis (median 8 mg/I, range 0-260 mg/I). No urinary C3 was detected in three of the patients who were in total remission. Urinary C3 was present in 12 patients who had significant proteinuria. Eight of the nine cases of mesangiocapillary glomerulonephritis showed detectable urinary C3. The median concentration was 17 mg/l and the range 0-52 mg/I. Six of the nine cases showed C3 deposits in glomerular capillary walls by immunofluorescence microscopy. None of the five cases of minimal lesion glomerulonephritis showed detectable urinary C3, but all had heavy proteinuria. None of the cases showed any C3 deposition by immunofluorescence microscopy.
Four cases of nephrotic syndrome due to amyloid disease proven by renal biopsy showed maximum urinary C3 concentrations of 6 mg/I, 8 mg/I, 10 mg/I, and 18 mg/I. In the group of non-immunological renal diseases there were cases of hydronephrosis (3), polycystic kidneys (2), analgesic nephropathy (1), chronic pyelonephritis (4), and renal tuberculosis (2). Five
of these 12 cases showed no detectable urinary C3; the median value was 5 mg/l and the range 0-8 mg/I. All cases had proteinuria greater than 1 g/l and were considered unlikely to have a glomerular lesion involving C3 fixation. In seven cases of proven urinary tract infection, no urinary C3 was detected. Urine samples from eight normal controls showed no urinary C3 even when concentrated up to 50 times. OF URINARY c3 FLUORESCENCE MICROSCOPY
RELATION
TO
IMMUNO-
Fifty-one patients were studied whose renal biopsies had been examined by immunofluorescence microscopy and from whom urine samples had been taken on the morning of, or within two days of, biopsy. Twenty-one of these showed positive immunofluorescence to C3 in glomerular capillary walls; 30 showed no glomerular C3. Of the 21 patients with glomerular C3 deposition, 17 showed detectable urinary C3. The median value was 9 mg/l and the range 0-75 mg/I. Of the 30 patients with no glomerular C3, 20 showed no detectable urinary C3, and the range was 0-20 mg/I. This relationship between positive immunofluorescence to C3 in glomerular capillary walls and urinary C3 concentration is highly significant (Wilcoxon Rank Sum Test 2a < 0-01; Student t test P < 0-001). RELATION OF PROTEINURIA
URINARY
c3
TO
DEGREE OF
A sample of fresh urine from 82 patients was tested for total protein. The correlation between this value and the C3 concentration in each sample was examined. A one-way analysis of variance showed no significant relationship (F = 2-0695; for 5% significance F must be > 5 67). Six patients had detectable urinary C3 in the absence of proteinuria; 15 patients had proteinuria greater than 3 g/l but no detectable urinary C3. RELATION OF URINARY OF PROTEINURIA
C3 TO SELECTIVITY
The urinary C3 concentration and the total protein concentration were determined in single urine samples from 24 patients, 12 of whom had selective proteinuria (k2 < 1-3) and 12 of whom had non-selective proteinuria (k2 > 2 3). All samples had a total protein concentration of over 1 g/l. The urinary C3 concentrations in the patients with selective and non-selective proteinuria were compared using a Wilcoxon Rank Sum Test; this showed that there was no significant difference in urinary C3 excretion between the two groups (2oc = > 010).
Significance ofurinary C3 excretion in glomerulonephritis Discussion C3 was the most suitable component for this study for four reasons: (1) it correlates well with whole complement levels in many situations; (2) it is involved in both the classical and the alternate pathways of complement activation; (3) a single complex of the earlier complement components can activate a large number of C3 molecules, so that changes in C3 concentration are more likely to be detectable than changes in the levels of earlier components; and (4) a suitable commercial antiserum is available. Although it would be of interest to know which fragments of C3 are present in urine and in what proportions, it is considered that, in this type of study, valid use may be made of an antiserum reacting with two or more of the major fragments. C3 has been detected in the urine of 59 of the 98 cases of renal disease studied. This is a higher proportion than has previously been reported. Lagrue et al (1969) found urinary C3 in nine of 24 cases, Gotoff et al (1965) in nine of 32 cases, and Ogg et al (1968) in one of 25 hypocomplementaemic patients. Of the three possible sources of urinary C3-(1) filtration from plasma, with other plasma proteins, through a damaged glomerular capillary basement membrane, (2) complement activation due to general inflammation within the urinary tract, (3) complement activation by immune complexes situated on the glomerular capillary basement membrane-we favour the third possibility. There is no correlation between the urinary C3 concentration and the total amount of protein in the urine. Detectable C3 may be present when proteinuria is absent, while in some patients with very heavy proteinuria (up to 10 g/l) no urinary C3 is detected. There is no relationship between urinary C3 concentration and the selectivity of proteinuria. Since C3 is a large molecule (mol wt 180 000), higher concentrations of C3 in the urine would be expected in cases of non-selective than selective proteinuria if urinary C3 arose by simple filtration from plasma, and this does not in fact occur. Since urinary C3 concentrations are not related to the degree or selectivity of proteinuria, it is unlikely that C3 in urine is derived to a significant extent from simple filtration through the glomerular capillary basement membrane. We have also found that urinary C3 is not detected in cases of proven urinary tract infection. It therefore seems likely that the third possible source of urinary C3, ie, complement activation by immune complexes on the glomerular capillary basement membrane, is the most significant. Supporting evidence for this is provided by the close
605
relationship between the detection of C3 in glomerular capillary walls by immunofluorescence microscopy and the detection of C3 in the urine. Since complement deposition in glomerular capillary walls is due to immune processes, this relationship suggests a similar origin for urinary C3. This view is also supported by the absence of urinary C3 in five cases with heavy proteinuria due to minimal lesion glomerulonephritis, in which glomerular deposition of immunoglobulins or complement cannot be detected by available methods, and by the observation that urinary C3 is undetectable or present in low concentrations in patients with proteinuria associated with non-immunological diseases such as polycystic renal disease or hydronephrosis. The detection of urinary C3 in four cases of nephrotic syndrome due to amyloid disease is compatible with this theory. It has been shown that amyloid in some cases contains fragments of immunoglobulins (Glenner et al, 1971a, b), and it has been suggested that a specific immunological mechanism is involved in the pathogenesis (Isobe and Osserman, 1974), possibly involving complement (Lachmann et al, 1962; Franklin and Zucker-Franklin, 1972). Urinary C3 excretion was found to bear some relationship to histological diagnosis. No urinary C3 was detected in cases of non-progressive proliferative glomerulonephritis and minimal lesion glomerulonephritis; low or undetectable concentrations were found in cases of non-immunological renal disease. Detectable urinary C3 concentrations were, however, found in almost all cases of progressive proliferative and rapidly progressive, membranous and mesangiocapillary glomerulonephritis, and in renal amyloidosis. The greatest range of concentrations was observed in progressive proliferative and in membranous glomerulonephritis. The absence of urinary C3 in cases of minimal lesion glomerulonephritis and non-immunological renal diseases might be due to the absence of complementfixing immune complexes in the glomeruli of these cases. In proliferative glomerulonephritis, immune complexes are frequently involved in the pathogenesis. Four of our cases of non-progressive proliferative glomerulonephritis showed deposits of immunoglobulin and C3 in glomerular capillary walls but had no urinary C3; a possible explanation is that urinary C3 excretion is very closely related in time to glomerular disease activity. If, as we have suggested above, urinary C3 arises from complement fixation by glomerular immune complexes, one would expect urinary C3 excretion to coincide with periods of active deposition of immune complexes in glomerular capillary walls. In the four cases mentioned above, the C3 deposits seen by immunofluorescence microscopy may have been derived
606AV
A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
from a previous episode of complement-fixing glomerular disease activity during which urinary C3 was present, but which had ceased by the time of biopsy, as had urinary C3 excretion. Our results give further evidence for this close relationship between urinary C3 concentration and current disease activity. In renal lesions characterized by continuing disease activity; such as progressive proliferative or membranous glomerulonephritis, urinary C3 was an almost constant finding. Significantly, the three cases of membranous glomerulonephritis with no urinary C3 were found to be in clinical remission, whereas the highest concentrations were found in patients with progressing disease. Long-term serial urine and serum C3 estimations were performed in a patient with proliferative glomerulonephritis associated with influenza virus A infection and in a patient with malignancy-associated nephrotic syndrome, in both of whom the disease eventually resolved. Both initially had low serum C3 and high urinary C3 concentrations. In both cases, urinary C3 ceased to be detectable at a very early stage, preceding by several weeks the return of serum C3 concentrations to normal and the disappearance of proteinuria. These findings suggest that urinary C3 estimations may provide a sensitive index of current glomerular disease activity. Urinary C3 estimation has many potential applications. It may be used in the diagnosis of renal disease, as an indicator of current glomerular disease activity, to assess patients' response to therapy, and to study the natural history of different types of glomerulonephritis.
Financial support for this study has been given by the Lawson Tait Medical and Scientific Trust, the Scottish Home and Health Department, and the Scottish Hospital Endowments Research Trust. We are grateful to Dr Anne T. Lambie and Dr A. Doig, Medical Renal Unit, Royal Infirmary, Edinburgh, Dr J. L. Anderton, Western General Hospital, Edinburgh, Dr W. S. Uttley, Royal Hospital for Sick Children, Edinburgh, and Dr H. Murray, Edinburgh University Student Health Centre, for permission to study patients under their care. We wish to thank Professor A. R. Currie for his advice and support, Dr Elizabeth Robertson, Blood Transfusion Service, Royal Infirmary, Edinburgh, for provision of normal sera, and Dr D. E. M. Taylor, Department of Physiology, University of Edinburgh, for advice on statistical analysis. We are grateful for the technical assistance of Mr J. K. Rae. References
Bokisch, V. A., Muller-Eberhard, H. J., and Cochrane, C. G.
(1969). Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. J. exp. Med., 129, 1109-1130. Franklin, E. C. and Zucker-Franklin, D. (1972). Current concepts of amyloid. Advanc. Intmunol., 15, 249-304. Glenner, G. G., Ein, D., Eanes, E. D., Bladen, H. A., Terry, W., and Page, D. L. (1971 a). Creation of 'amyloid' fibrils from Bence-Jones proteins in vitro. Science, 174, 712-714. Glenner, G. G., Terry, W., Harada, M., Isersky, C., and Page, D. (1971b). Amyloid fibril proteins: proof of homology with immunoglobulin light chains by sequence analyses. Science, 172, 1150-1151. Gotoff, S. P., Fellers, F. X., Vawter, G. F., Janeway, C. A., and Rosen, F. S. (1965). The gic globulin in childhood nephrotic syndrome. New Engl. J. Med., 273, 524-529. Gutman, R. A., Striker, G. E., Gilliland, B. C., and Cutler, R. E. (1972). The immune complex glomerulonephritis of bacterial endocarditis. Medicine (Baltimore), 51, 1-25. Hoq, M. S., Anderton, J. L., Cunningham, M., and Cash, J. D. (1974). Urinary excretion of fibrinogen-related materials, complement, and immunoglobulins in proliferative glomerulonephritis and after renal transplantation. Brit. med. J., 2, 535-538. Isobe, T. and Osserman, E. F. (1974). Patterns of amyloidosis and their association with plasma-cell dyscrasia, monoclonal immunoglobulins and Bence-Jones proteins. NewS Engl. J. Med., 290, 473-477. Klemperer, M. R., Gotoff, S. P., Alper, C. A., Levin, A. S., and Rosen, F. S. (1965). Estimation of the serum ic globulin concentration: its relation to the serum hemolytic complement titer. Pediatrics, 35, 765-769. Koffler, D., Agnello, V., Thoburn, R., and Kunkel, H. G. (1971). Systemic lupus erythematosus; prototype of immune complex nephritis in man. J. exp. Med., 134, 169S-1 79S. Kohler, P. F. and Ten Bensel, R. (1969). Serial complement component alterations in acute glomerulonephritis and systemic lupus erythematosus. Clin. exp. Immunol., 4, 191-202. Lachmann, P. J., Muller-Eberhard, H. J., Kunkel, H. G., and Paronetto, F. (1962). The localisation of in vivo bound complement in tissue sections. J. exp. Med., 115, 63-82. Lagrue, G., Brecy, H., and Hartmann, L. (1969). La complementurie dans les glomerulopathies humaines. Rev. franf. Etud. clin. biol., 14, 346-353. Lange, K., Graig, F., Oberman, J., Slobody, L., Ogur, G., and LoCasto, F. (1951). Changes in serum complement during the course and treatment of glomerulonephritis. Arch. intern. Med., 88, 433-445. Lange, K., Wasserman, E., and Slobody, L. B. (1960). The significance of serum complement levels for the diagnosis and prognosis of acute and subacute glomerulonephritis and lupus erythematosus disseminatus. Ann. intern. Med., 53, 636-646. Lange, K. and Wenk, E. J. (1954). Complement components in the sera and urines of patients with severe proteinurias. Amer. J. med. Sci., 228, 448-453. Lundh, B., Hedberg, H., and Laurell, A. B. (1970). Studies of the third component of complement in synovial fluid from arthritic patients. I. Immunochemical quantitation and relation to total complement. Clin. exp. Immuinol., 6 407-411. MacLean, P. R., and Robson, J. S. (1967). A simple method for determining selectivity of proteinuria. Lancet, 1, 539-542. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235-254.
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Significance of urinary C3 excretion in glomerulonephritis Ogg, C. S., Cameron, J. S., and White, R. H. R. (1968). The C'3 component of complement (Pic-globulin) in patients with heavy proteinuria. Lancet, 2, 78-81. Ruddy, S., Gigli, I., and Austen, K. F. (1972). The complement system of man (I). New Engl. J. Med., 287, 489-495. Vallota, E. H., Forristal, J., Spitzer, R. E., Davis, N. C., and West, C. D. (1970) .Characteristics of a non-complement dependent C3-reactive complex formed from factors in nephritic and normal serum. J. exp. Med., 131, 1306-1324. West, C. D, Davis, N. C., Forristal, J, Herbst, J., and Spitzer, R. (1966). Antigenic determinants of human PIc- and PIG-globulins. J. Immunol., 96, 650-658.
West, C. D., Northway, J. D., and Davis, N. C. (1964). Serum levels of Pic globulin, a complement component, in the nephritides, lipoid nephrosis, and other conditions. J. clin. Invest., 43, 1507-1517. Williams, B. D., Clarkson, A. R., Row, P. G., Groves, R. J., and Cameron, J. S. (1974). Urinary excretion of C3 antigen in glomerulonephritis. Brit. med. J., 4, 21-23.
Requests for reprints should be addressed to Dr D. Thomson, Department of Pathology, University Medical School, Teviot Place, Edinburgh, EH8 9AG.
The June 1976 Issue THE JUNE 1976 ISSUE CONTAINS THE FOLLOWING PAPERS
The history of haemophilia G. I. C. INGRAM
Tests of performance of anaerobic jars B. WATT, J. G. COLLEE, AND R. BROWN
Hepatitis in clinical laboratories 1973-74 N. R. GRIST
Anticonnective tissue and other antitissue 'antibodies' in the sera of patients with coeliac disease compared with the findings in a mixed hospital
Isolation of oxidase-positive Gram-negative cocci not belonging to the genus Neisseria from the urogenital tract D. J. PLATT AND J. J. S. SNELL
population N. WILLIAMSON, P. ASQUITH, P. L.
Computer printing and ffling of microbiology reports 1 Description of the system C. S. GOODWIN AND B.
STOKES, A. W. JOWETT, AND W. T. COOKE
CLARE SMITH
A rapidly produced 1251 labelled autologous fibrinogen: in vitro properties and preliminary metabolic studies in man R. J. HAWKER AND LINDA M. HAWKER
Computer printing and filing of microbiology reports 2 Evaluation and comparison with a manual system, and comparison of two manual systems c. s. GOODWIN
Iliac bone marrow mast cells in relation to the renal osteodystrophy of patients treated by haemodialysis H. A. ELLIS AND KATHLEEN M. PEART
Serum urea analysis using the Beckman BUN Analyzer K. S. CHUA AND I. K. TAN
Technical methods A simple radio immunoassay for serum /3-oestriol in pregnancy D. WATSON
M. G. NICKLIN, J. V. HEWITT, AND
Spindle-cell tumours and hypoglycaemia P. R.
Morpholigical methods in the study of the gut immune system in man J. M. SKINNER AND R.
MILLARD, D. W. JERROME, AND G. H. MILLWARDSADLER
WHITEHEAD
Comparative study of two systems for detecting bacteraemia and septicaemia N. G. WOOD
Letter to the Editor
Book Review
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Significance of urinary C3 excretion in glomerulonephritis A. D. CUMMING, D. THOMSON, A. M. DAVISON, AND J. S. ROBSON From the Department of Pathology and the Medical Renal Unit, Department of Medicine, University of Edinburgh and the Royal Infirmary, Edinburgh EH3 9 YW
The third component of complement (C3) was measured in the urine of 98 patients with variety of renal diseases. Renal biopsy was performed on 83 of the patients and examined by light, electron, and immunofluorescence microscopy. Urinary C3 was detected in cases of membranous glomerulonephritis, mesangiocapillary glomerulonephritis, rapidly progressive glomerulonephritis, and renal amyloidosis. It was not detected in minimal lesion glomerulonephritis; in cases of proliferative glomerulonephritis it was detected only in those showing histological evidence of a progressive lesion. Concentrations were low or undetectable in cases of non-immunological renal diseases. There was a good correlation between urinary C3 concentrations and the deposition of C3 in glomerular capillary walls, as seen by immunofluorescence microscopy, and there was no correlation with the degree or selectivity of proteinuria. Urinary C3 excretion appears to be an accurate indicator of continuing activity of disease. It is suggested that the presence of C3 in urine is due to complement fixation by immune complexes in glomerular capillary walls, and that urinary C3 estimations have potential applications in the study of glomerulonephritis.
SYNOPSIS a
The association of complement with renal disease has been known since 1902 when Hedinger described low serum complement in 'acute nephritis'. Many studies have subsequently shown that low serum complement titres are characteristic of acute post-
information about renal immune processes. Lange and Wenk (1954) investigated urinary loss of complement as a possible cause of low serum whole complement titres in glomerulonephritis and found that there was no correlation between the urinary streptococcal glomerulonephritis, mesangiocapillary complement titre and the serum complement titre, glomerulonephritis, systemic lupus erythematosus or the degree of proteinuria. Gotoff et al (1965) with renal involvement, and nephritis accompanying found C3 in the urine of patients with nephrotic subacute bacterial endocarditis (Lange et al, 1951, syndrome with heavy proteinuria but not in cases 1960; West et al, 1964; Gotoff et al, 1965; Ogg et al, of acute or progressive glomerulonephritis. Ogg et al 1968; Gutman et al, 1972). This is thought to be (1968) found urinary C3 in only one of 25 patients due in many cases to utilization of complement by with low serum C3 concentrations. immune complexes (Kohler and Ten Bensel, 1969; Lagrue et al (1969) examined complement activity Koffler et al, 1971), although direct activation of the in the urine of 24 patients with glomerulonephritis third component by the alternate pathway is known and found complement components in the urine of to occur in some types of glomerulonephritis nine patients. Complement excretion did not (Vallota et al, 1970). It has also been shown that in correlate with the degree or selectivity of proteinuria many situations there is a correlation between the but varied in the four types of glomerulonephritis whole complement titre and the concentration of studied. They concluded that the mechanism of the third component of complement (C3) (Klem- urinary complement excretion remained to be determined and that its frequency and significance perer et al, 1965; Lundh et al, 1970). Since complement is activated during immune should be further studied. Hoq et al (1974) studied reactions, it seems possbile that examination of 15 cases of proliferative glomerulonephritis and 10 complement excretion in the urine could give cases after renal homotransplantation. Urinary C3 concentrations correlated with urinary concentrations of fibrin degradation products and heteroReceived for publication 3 December 1975 601
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A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
phile haemagglutinins, were not related to total urinary protein, and gave information on response to treatment and occurrence of rejection. Williams et al (1974) studied 123 patients with glomerulonephritis; urinary C3 was detected often in mesangiocapillary and membranous glomerulonephritis and in focal glomerulosclerosis, less frequently in lupus nephritis and a mixed group of cases with proliferative glomerulonephritis, and rarely in minimal lesion glomerulonephritis. The present study examines the concentration and significance of C3 in the urine of patients with renal disease. Immunofluorescence microscopy has demonstrated the presence of material, consisting of immunoglobulins and complement components, within the glomerular capillary wall in several forms of glomerulonephritis, and it seems likely that deposition of such material could give rise to increased components of complement in the urine. This was investigated by examining (1) the relationship of urinary C3 to the type of glomerulonephritis present, and also urinary C3 concentrations in cases of proteinuria thought not to be due to acomplementfixing glomerular immune reaction; (2) the relationship between urinary C3 concentrations and C3 deposition in glomerular capillary walls detected by immunofluorescence microscopy; (3) the relationship between urinary C3 concentrations and the degree and selectivity of proteinuria.
and a raised ASO titre. In 11 cases there were no polymorphs in the glomeruli, there was slight to moderate hypercellularity of mesangial regions, and these were considered to be cases of mild proliferative glomerulonephritis. Twenty-two cases showed enlarged, hyaline, and hypercellular mesangial regions, producing variable degrees of hyalinization of the tufts, and in some cases capsular adhesions and small epithelial crescents were additional features of note. These changes were considered to indicate progression of the disease process resulting in some permanent glomerular damage. They were therefore designated as cases of proliferative glomerulonephritis showing histological evidence of progression. There were six cases of rapidly progressive glomerulonephritis, the main histological features being florid circumferential epithelial crescents in most glomeruli, compressing the capillary tufts, with little or no mesangial hypercellularity. Other types of renal disease were also diagnosed histologically according to conventional criteria, except for eight cases of non-immunological renal disease and seven cases of urinary tract infection, which were diagnosed on clinical grounds since a biopsy had not been performed. HISTOLOGICAL METHODS
All biopsies were examined by light microscopy; electron microscopy was performed in 64 cases and immunofluorescence microscopy in 51. For light Material and Methods microscopy, biopsy material was fixed in corrosive formol saline for 18-24 hours, sectioned at 2 ,um, PATIENTS STUDIED The concentration of urinary C3 was measured in and stained by the haematoxylin and eosin, picro 98 patients, 83 of whom underwent renal biopsy. Mallory, periodic-acid Schiff, and Martius scarlet The histological diagnoses are shown in the table. blue methods. For electron microscopy the tissue Of the 18 cases of proliferative glomerulonephritis was fixed in 1 % osmium tetroxide, sometimes with without histological evidence of progression, seven preliminary glutaraldehyde fixation. It was dehydshowed an acute, exudative lesion. They were rated in graded alcohols and embedded in Araldite histologically similar to classical poststreptococcal or Epon. Sections were cut on a Porter Blum MT2 glomerulonephritis, showing diffuse enlargement of ultratome at 50 nm, stained with lead citrate and tufts, moderate to marked mesangial hypercellularity, uranyl acetate, and viewed in an AEI EM6 electron and a polymorphonuclear infiltrate. Two of these microscope. For immunofluorescence microscopy cases had a proven previous streptococcal infection fresh tissue was 'snap frozen' with Drikold and sectioned at 2 to 3 ,um in a cryostat. After fixation with 95 % alcohol for 10 minutes, sections were No. of Cases Type of Disease incubated with fluorescein isothiocyanate conjugated (FITC) antihuman serum to C3, IgG, IgA, IgM, and Proliferative glomerulonephritis 18 no histological evidence of progression fibrin-fibrinogen (Hoechst). Sections were viewed 22 histological evidence of progression in a Leitz Ortholux microscope using an HBO 200 6 Rapidly progressive glomerulonephritis 15 Membranous glomerulonephritis with BG12 and BG38 primary filters and a lamp 9 Mesangiocapillary glomerulonephritis K510 barrier filter. 5 Minimal lesion glomerulonephritis Renal amyloidosis Non-immunological renal disease Urinary tract infection
4 12 7
Table Diagnoses and number ofpatients of each type
URINARY c3 AND SELECTIVITY
For C3 estimations 10-30 ml samples of urine were used; where possible these were early morning
Significance of urinary C3 excretion in glomerulonephritis
603
obtained. Four serial dilutions of a pool of 30 normal human sera were included on each plate. The C3 concentration of this pool was measured using standard sera (Travenol Laboratories) and found to be 1-3 g/l. Results of test samples were expressed as mg/l. A single sample was tested on 20 separate occasions and the standard deviation was found to be ± 8 %. Protein selectivity was estimated by the method of MacLean and Robson (1967).
specimens. Three to four daily samples were obtained from most patients and the highest urinary C3 concentration found in each patient was used in subsequent analysis. Samples were tested for total protein, using Albustix (Ames), dialysed against running tap water for 8 hours, and left overnight in a solution of polyethylene glycol (Carbowax) at - 40C, which concentrated each sample 10-15 times. Samples were stored at -30°C until they were tested. C3 concentrations were measured by single radial immunodiffusion; plates were prepared using a modification of the method of Mancini (Mancini et al, 1965). 0 3 ml of rabbit antiserum to human /IC-PIA globulin (Hoechst) was used in each 16-well plate. We have not ascertained with which fragments of C3 the antiserum reacts; previous work suggests it may react with intact C3, C3b, and C3c (West et al, 1966; Bokisch et al, 1969; Ruddy et al, 1972). The antiserum was used in immunoelectrophoresis of aged human serum; a single arc was
Results RELATION
OF
URINARY
c3
TO HISTOLOGY
(FIGURE) Urinary C3 was not detectable in any of the 18 patients with proliferative glomerulonephritis who showed no histological evidence of progression. Of the seven patients in this group with an acute exudative lesion, all had moderate or heavy proteinuria and three showed immunofluorescence to C3 in glomerular capillary walls. Nine of the 11
TYPE OF RENAL DISEASE
Proliferative Glonierulonephritis - no histological evidence of progression
Proliferative Glonierulonephritis
-
histological evidence of progression
0'#
Rapidly Progressive Glomerulonephritis
.
s
Membranous Glomerulonephritis
1*00i *0 em
Mesangiocapillary Glomerulonephritis
*
0
C
0
*
I
me *
I
0
I
G*~~~~~~~~~~
Minimal Lesion Glomerulonephritis
00n Renal Amyloidosis
Non-Immunological Renal Diseases Urinary Tract Infection
10
0
20
30
40
Figure Maximum concentrations of C3 in urine from patients with renal disease. Conversion: SI to Traditional Units-C3: I
50
URINARY C3 CONCENTRATION * DETECTABLE NOT DETECTABLE
mg/li = 0.1 mg/100 ml
60
mg/l)
70
80 100
260
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A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
patients with mild proliferative glomerulonephritis showed significant proteinuria and one showed some C3 deposition in glomerular capillary walls by immunofluorescence microscopy. In follow-up observations between 13 months and five years after onset of the disease, one of the 18 patients had raised plasma urea and creatinine concentrations, and one had recurrent proteinuria. The other patients were symptom-free, had normal plasma urea and creatinine concentrations, normal blood pressure, and no proteinuria or haematuria. In contrast, 21 of the 22 cases of progressive proliferative glomerulonephritis showed significant urinary C3 concentrations (median 10 mg/I, range 0-210 mg/i). Of the 12 cases studied by immunofluorescence microscopy, five showed immunofluorescence to C3 in glomerular capillary walls. In follow-up studies between one and 11 years after onset of the disease, all but one of the 22 patients showed one or more of the following abnormalitiesraised plasma urea or creatinine concentration, raised blood pressure, persistent proteinuria or haematuria. The patient whose disease clinically resolved had a maximum urinary C3 concentration of 10 mg/l. The six cases of rapidly progressive glomerulonephritis all showed detectable urinary C3, but, in comparison with the progressive proliferative cases, a smaller range of concentrations was found (median 12 mg/I, range 8-25 mg/i). Four cases showed immunofluorescence to C3 in glomeruli. The greatest range of urinary C3 concentrations was found in the 15 cases of membranous glomerulonephritis (median 8 mg/I, range 0-260 mg/I). No urinary C3 was detected in three of the patients who were in total remission. Urinary C3 was present in 12 patients who had significant proteinuria. Eight of the nine cases of mesangiocapillary glomerulonephritis showed detectable urinary C3. The median concentration was 17 mg/l and the range 0-52 mg/I. Six of the nine cases showed C3 deposits in glomerular capillary walls by immunofluorescence microscopy. None of the five cases of minimal lesion glomerulonephritis showed detectable urinary C3, but all had heavy proteinuria. None of the cases showed any C3 deposition by immunofluorescence microscopy.
Four cases of nephrotic syndrome due to amyloid disease proven by renal biopsy showed maximum urinary C3 concentrations of 6 mg/I, 8 mg/I, 10 mg/I, and 18 mg/I. In the group of non-immunological renal diseases there were cases of hydronephrosis (3), polycystic kidneys (2), analgesic nephropathy (1), chronic pyelonephritis (4), and renal tuberculosis (2). Five
of these 12 cases showed no detectable urinary C3; the median value was 5 mg/l and the range 0-8 mg/I. All cases had proteinuria greater than 1 g/l and were considered unlikely to have a glomerular lesion involving C3 fixation. In seven cases of proven urinary tract infection, no urinary C3 was detected. Urine samples from eight normal controls showed no urinary C3 even when concentrated up to 50 times. OF URINARY c3 FLUORESCENCE MICROSCOPY
RELATION
TO
IMMUNO-
Fifty-one patients were studied whose renal biopsies had been examined by immunofluorescence microscopy and from whom urine samples had been taken on the morning of, or within two days of, biopsy. Twenty-one of these showed positive immunofluorescence to C3 in glomerular capillary walls; 30 showed no glomerular C3. Of the 21 patients with glomerular C3 deposition, 17 showed detectable urinary C3. The median value was 9 mg/l and the range 0-75 mg/I. Of the 30 patients with no glomerular C3, 20 showed no detectable urinary C3, and the range was 0-20 mg/I. This relationship between positive immunofluorescence to C3 in glomerular capillary walls and urinary C3 concentration is highly significant (Wilcoxon Rank Sum Test 2a < 0-01; Student t test P < 0-001). RELATION OF PROTEINURIA
URINARY
c3
TO
DEGREE OF
A sample of fresh urine from 82 patients was tested for total protein. The correlation between this value and the C3 concentration in each sample was examined. A one-way analysis of variance showed no significant relationship (F = 2-0695; for 5% significance F must be > 5 67). Six patients had detectable urinary C3 in the absence of proteinuria; 15 patients had proteinuria greater than 3 g/l but no detectable urinary C3. RELATION OF URINARY OF PROTEINURIA
C3 TO SELECTIVITY
The urinary C3 concentration and the total protein concentration were determined in single urine samples from 24 patients, 12 of whom had selective proteinuria (k2 < 1-3) and 12 of whom had non-selective proteinuria (k2 > 2 3). All samples had a total protein concentration of over 1 g/l. The urinary C3 concentrations in the patients with selective and non-selective proteinuria were compared using a Wilcoxon Rank Sum Test; this showed that there was no significant difference in urinary C3 excretion between the two groups (2oc = > 010).
Significance ofurinary C3 excretion in glomerulonephritis Discussion C3 was the most suitable component for this study for four reasons: (1) it correlates well with whole complement levels in many situations; (2) it is involved in both the classical and the alternate pathways of complement activation; (3) a single complex of the earlier complement components can activate a large number of C3 molecules, so that changes in C3 concentration are more likely to be detectable than changes in the levels of earlier components; and (4) a suitable commercial antiserum is available. Although it would be of interest to know which fragments of C3 are present in urine and in what proportions, it is considered that, in this type of study, valid use may be made of an antiserum reacting with two or more of the major fragments. C3 has been detected in the urine of 59 of the 98 cases of renal disease studied. This is a higher proportion than has previously been reported. Lagrue et al (1969) found urinary C3 in nine of 24 cases, Gotoff et al (1965) in nine of 32 cases, and Ogg et al (1968) in one of 25 hypocomplementaemic patients. Of the three possible sources of urinary C3-(1) filtration from plasma, with other plasma proteins, through a damaged glomerular capillary basement membrane, (2) complement activation due to general inflammation within the urinary tract, (3) complement activation by immune complexes situated on the glomerular capillary basement membrane-we favour the third possibility. There is no correlation between the urinary C3 concentration and the total amount of protein in the urine. Detectable C3 may be present when proteinuria is absent, while in some patients with very heavy proteinuria (up to 10 g/l) no urinary C3 is detected. There is no relationship between urinary C3 concentration and the selectivity of proteinuria. Since C3 is a large molecule (mol wt 180 000), higher concentrations of C3 in the urine would be expected in cases of non-selective than selective proteinuria if urinary C3 arose by simple filtration from plasma, and this does not in fact occur. Since urinary C3 concentrations are not related to the degree or selectivity of proteinuria, it is unlikely that C3 in urine is derived to a significant extent from simple filtration through the glomerular capillary basement membrane. We have also found that urinary C3 is not detected in cases of proven urinary tract infection. It therefore seems likely that the third possible source of urinary C3, ie, complement activation by immune complexes on the glomerular capillary basement membrane, is the most significant. Supporting evidence for this is provided by the close
605
relationship between the detection of C3 in glomerular capillary walls by immunofluorescence microscopy and the detection of C3 in the urine. Since complement deposition in glomerular capillary walls is due to immune processes, this relationship suggests a similar origin for urinary C3. This view is also supported by the absence of urinary C3 in five cases with heavy proteinuria due to minimal lesion glomerulonephritis, in which glomerular deposition of immunoglobulins or complement cannot be detected by available methods, and by the observation that urinary C3 is undetectable or present in low concentrations in patients with proteinuria associated with non-immunological diseases such as polycystic renal disease or hydronephrosis. The detection of urinary C3 in four cases of nephrotic syndrome due to amyloid disease is compatible with this theory. It has been shown that amyloid in some cases contains fragments of immunoglobulins (Glenner et al, 1971a, b), and it has been suggested that a specific immunological mechanism is involved in the pathogenesis (Isobe and Osserman, 1974), possibly involving complement (Lachmann et al, 1962; Franklin and Zucker-Franklin, 1972). Urinary C3 excretion was found to bear some relationship to histological diagnosis. No urinary C3 was detected in cases of non-progressive proliferative glomerulonephritis and minimal lesion glomerulonephritis; low or undetectable concentrations were found in cases of non-immunological renal disease. Detectable urinary C3 concentrations were, however, found in almost all cases of progressive proliferative and rapidly progressive, membranous and mesangiocapillary glomerulonephritis, and in renal amyloidosis. The greatest range of concentrations was observed in progressive proliferative and in membranous glomerulonephritis. The absence of urinary C3 in cases of minimal lesion glomerulonephritis and non-immunological renal diseases might be due to the absence of complementfixing immune complexes in the glomeruli of these cases. In proliferative glomerulonephritis, immune complexes are frequently involved in the pathogenesis. Four of our cases of non-progressive proliferative glomerulonephritis showed deposits of immunoglobulin and C3 in glomerular capillary walls but had no urinary C3; a possible explanation is that urinary C3 excretion is very closely related in time to glomerular disease activity. If, as we have suggested above, urinary C3 arises from complement fixation by glomerular immune complexes, one would expect urinary C3 excretion to coincide with periods of active deposition of immune complexes in glomerular capillary walls. In the four cases mentioned above, the C3 deposits seen by immunofluorescence microscopy may have been derived
606AV
A. D. Cumming, D. Thomson, A. M. Davison, and J. S. Robson
from a previous episode of complement-fixing glomerular disease activity during which urinary C3 was present, but which had ceased by the time of biopsy, as had urinary C3 excretion. Our results give further evidence for this close relationship between urinary C3 concentration and current disease activity. In renal lesions characterized by continuing disease activity; such as progressive proliferative or membranous glomerulonephritis, urinary C3 was an almost constant finding. Significantly, the three cases of membranous glomerulonephritis with no urinary C3 were found to be in clinical remission, whereas the highest concentrations were found in patients with progressing disease. Long-term serial urine and serum C3 estimations were performed in a patient with proliferative glomerulonephritis associated with influenza virus A infection and in a patient with malignancy-associated nephrotic syndrome, in both of whom the disease eventually resolved. Both initially had low serum C3 and high urinary C3 concentrations. In both cases, urinary C3 ceased to be detectable at a very early stage, preceding by several weeks the return of serum C3 concentrations to normal and the disappearance of proteinuria. These findings suggest that urinary C3 estimations may provide a sensitive index of current glomerular disease activity. Urinary C3 estimation has many potential applications. It may be used in the diagnosis of renal disease, as an indicator of current glomerular disease activity, to assess patients' response to therapy, and to study the natural history of different types of glomerulonephritis.
Financial support for this study has been given by the Lawson Tait Medical and Scientific Trust, the Scottish Home and Health Department, and the Scottish Hospital Endowments Research Trust. We are grateful to Dr Anne T. Lambie and Dr A. Doig, Medical Renal Unit, Royal Infirmary, Edinburgh, Dr J. L. Anderton, Western General Hospital, Edinburgh, Dr W. S. Uttley, Royal Hospital for Sick Children, Edinburgh, and Dr H. Murray, Edinburgh University Student Health Centre, for permission to study patients under their care. We wish to thank Professor A. R. Currie for his advice and support, Dr Elizabeth Robertson, Blood Transfusion Service, Royal Infirmary, Edinburgh, for provision of normal sera, and Dr D. E. M. Taylor, Department of Physiology, University of Edinburgh, for advice on statistical analysis. We are grateful for the technical assistance of Mr J. K. Rae. References
Bokisch, V. A., Muller-Eberhard, H. J., and Cochrane, C. G.
(1969). Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. J. exp. Med., 129, 1109-1130. Franklin, E. C. and Zucker-Franklin, D. (1972). Current concepts of amyloid. Advanc. Intmunol., 15, 249-304. Glenner, G. G., Ein, D., Eanes, E. D., Bladen, H. A., Terry, W., and Page, D. L. (1971 a). Creation of 'amyloid' fibrils from Bence-Jones proteins in vitro. Science, 174, 712-714. Glenner, G. G., Terry, W., Harada, M., Isersky, C., and Page, D. (1971b). Amyloid fibril proteins: proof of homology with immunoglobulin light chains by sequence analyses. Science, 172, 1150-1151. Gotoff, S. P., Fellers, F. X., Vawter, G. F., Janeway, C. A., and Rosen, F. S. (1965). The gic globulin in childhood nephrotic syndrome. New Engl. J. Med., 273, 524-529. Gutman, R. A., Striker, G. E., Gilliland, B. C., and Cutler, R. E. (1972). The immune complex glomerulonephritis of bacterial endocarditis. Medicine (Baltimore), 51, 1-25. Hoq, M. S., Anderton, J. L., Cunningham, M., and Cash, J. D. (1974). Urinary excretion of fibrinogen-related materials, complement, and immunoglobulins in proliferative glomerulonephritis and after renal transplantation. Brit. med. J., 2, 535-538. Isobe, T. and Osserman, E. F. (1974). Patterns of amyloidosis and their association with plasma-cell dyscrasia, monoclonal immunoglobulins and Bence-Jones proteins. NewS Engl. J. Med., 290, 473-477. Klemperer, M. R., Gotoff, S. P., Alper, C. A., Levin, A. S., and Rosen, F. S. (1965). Estimation of the serum ic globulin concentration: its relation to the serum hemolytic complement titer. Pediatrics, 35, 765-769. Koffler, D., Agnello, V., Thoburn, R., and Kunkel, H. G. (1971). Systemic lupus erythematosus; prototype of immune complex nephritis in man. J. exp. Med., 134, 169S-1 79S. Kohler, P. F. and Ten Bensel, R. (1969). Serial complement component alterations in acute glomerulonephritis and systemic lupus erythematosus. Clin. exp. Immunol., 4, 191-202. Lachmann, P. J., Muller-Eberhard, H. J., Kunkel, H. G., and Paronetto, F. (1962). The localisation of in vivo bound complement in tissue sections. J. exp. Med., 115, 63-82. Lagrue, G., Brecy, H., and Hartmann, L. (1969). La complementurie dans les glomerulopathies humaines. Rev. franf. Etud. clin. biol., 14, 346-353. Lange, K., Graig, F., Oberman, J., Slobody, L., Ogur, G., and LoCasto, F. (1951). Changes in serum complement during the course and treatment of glomerulonephritis. Arch. intern. Med., 88, 433-445. Lange, K., Wasserman, E., and Slobody, L. B. (1960). The significance of serum complement levels for the diagnosis and prognosis of acute and subacute glomerulonephritis and lupus erythematosus disseminatus. Ann. intern. Med., 53, 636-646. Lange, K. and Wenk, E. J. (1954). Complement components in the sera and urines of patients with severe proteinurias. Amer. J. med. Sci., 228, 448-453. Lundh, B., Hedberg, H., and Laurell, A. B. (1970). Studies of the third component of complement in synovial fluid from arthritic patients. I. Immunochemical quantitation and relation to total complement. Clin. exp. Immuinol., 6 407-411. MacLean, P. R., and Robson, J. S. (1967). A simple method for determining selectivity of proteinuria. Lancet, 1, 539-542. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235-254.
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Significance of urinary C3 excretion in glomerulonephritis Ogg, C. S., Cameron, J. S., and White, R. H. R. (1968). The C'3 component of complement (Pic-globulin) in patients with heavy proteinuria. Lancet, 2, 78-81. Ruddy, S., Gigli, I., and Austen, K. F. (1972). The complement system of man (I). New Engl. J. Med., 287, 489-495. Vallota, E. H., Forristal, J., Spitzer, R. E., Davis, N. C., and West, C. D. (1970) .Characteristics of a non-complement dependent C3-reactive complex formed from factors in nephritic and normal serum. J. exp. Med., 131, 1306-1324. West, C. D, Davis, N. C., Forristal, J, Herbst, J., and Spitzer, R. (1966). Antigenic determinants of human PIc- and PIG-globulins. J. Immunol., 96, 650-658.
West, C. D., Northway, J. D., and Davis, N. C. (1964). Serum levels of Pic globulin, a complement component, in the nephritides, lipoid nephrosis, and other conditions. J. clin. Invest., 43, 1507-1517. Williams, B. D., Clarkson, A. R., Row, P. G., Groves, R. J., and Cameron, J. S. (1974). Urinary excretion of C3 antigen in glomerulonephritis. Brit. med. J., 4, 21-23.
Requests for reprints should be addressed to Dr D. Thomson, Department of Pathology, University Medical School, Teviot Place, Edinburgh, EH8 9AG.
The June 1976 Issue THE JUNE 1976 ISSUE CONTAINS THE FOLLOWING PAPERS
The history of haemophilia G. I. C. INGRAM
Tests of performance of anaerobic jars B. WATT, J. G. COLLEE, AND R. BROWN
Hepatitis in clinical laboratories 1973-74 N. R. GRIST
Anticonnective tissue and other antitissue 'antibodies' in the sera of patients with coeliac disease compared with the findings in a mixed hospital
Isolation of oxidase-positive Gram-negative cocci not belonging to the genus Neisseria from the urogenital tract D. J. PLATT AND J. J. S. SNELL
population N. WILLIAMSON, P. ASQUITH, P. L.
Computer printing and ffling of microbiology reports 1 Description of the system C. S. GOODWIN AND B.
STOKES, A. W. JOWETT, AND W. T. COOKE
CLARE SMITH
A rapidly produced 1251 labelled autologous fibrinogen: in vitro properties and preliminary metabolic studies in man R. J. HAWKER AND LINDA M. HAWKER
Computer printing and filing of microbiology reports 2 Evaluation and comparison with a manual system, and comparison of two manual systems c. s. GOODWIN
Iliac bone marrow mast cells in relation to the renal osteodystrophy of patients treated by haemodialysis H. A. ELLIS AND KATHLEEN M. PEART
Serum urea analysis using the Beckman BUN Analyzer K. S. CHUA AND I. K. TAN
Technical methods A simple radio immunoassay for serum /3-oestriol in pregnancy D. WATSON
M. G. NICKLIN, J. V. HEWITT, AND
Spindle-cell tumours and hypoglycaemia P. R.
Morpholigical methods in the study of the gut immune system in man J. M. SKINNER AND R.
MILLARD, D. W. JERROME, AND G. H. MILLWARDSADLER
WHITEHEAD
Comparative study of two systems for detecting bacteraemia and septicaemia N. G. WOOD
Letter to the Editor
Book Review
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